Microscopic evaluation of the immunohistochemically stained slides from primary PCa and PCa bone metastasis revealed membranous expression of E-cadherin; cytoplasmic expression of Vimentin and PDGF-D, and nuclear of NF-κB, Notch-1 and ZEB1 ( and ). The expression pattern of the markers varied according to the location within the tumor with high expression of the EMT phenotype at the invasive tumor front (ITF) compared to the center of the tumor cell clusters. The difference in the expression levels of EMT markers between PCa and PCa bone metastasis are detailed in and . Upon statistical analysis of the EMT markers' expression levels among the PCa and bone metastasis, using the Fisher's Exact test, if the intensity and % of positive cells were considered separately, only the % of positive Notch-1 cells were statistically significantly higher in the PCa bone metastasis (p=0.033). However when both intensity and % of positive cells were analyzed together to give a final score of high vs. low expression, then Notch-1 expression was found to be approaching statistical significance in bone metastatic PCa compared to primary PCa, suggesting its role in PCa bone metastasis (p=0.057) (). For the remaining markers there was no significant difference in the expression, intensity, % of positive cells and scores in PCa vs. PCa bone metastasis.
Figure 1 Photomicrophotographs of EMT markers: (A) E-cadherin in PCa showing membranous expression in the center of the tumor cell clusters (circle), and loss of expression at the invasive tumor front (arrow) × 400. (B) E-cadherin in bone metastatic PCa (more ...)
Figure 2 Photomicrographs of EMT markers: (A) PCa showing PDGF-D over-expression at the center of the tumor cell clusters (circle) and low expression at the invasive tumor front (arrows) × 400. (B) Bone metastatic PCa (arrow) showing variable PDGF-D expression (more ...)
Marker expression as intensity and percent of positive cells
EMT Marker expression levels in PCa & bone metastasis
Dot-plot graph showing Notch-1 expression in the primary and bone metastatic PCa.
Cancer is the cause of death in up to 25% of the population in the United States, and the vast majority of these are of epithelial cell origin [1
]. During carcinogenesis, the immotile, polarized epithelial cells acquire highly migratory, apolar fibroblast-like features through a transient phenomenon referred to as Epithelial-to-Mesenchymal Transition (EMT). This indispensable process empowers the epithelial cancer cells with invasive and metastatic capabilities through acquisition of molecular alterations characteristic of mesenchymal phenotypes. It is known that EMT is associated with altered expression of growth and transcription factors and modified expression of cytoskeletal and adhesion molecules[22
]. These can serve as potential targets for EMT for both cancer prevention and/or treatment. Inhibition of EMT, targeted killing of EMT phenotypic cells or the reversal of EMT phenotype, which will sensitize the cancer cells to conventional therapeutics are becoming a great promise for the treatment management of cancer patients.
Although EMT has been described in normal tissue development during organogenesis, during tissue remodeling and in wound healing, it is inappropriately reactivated during the development and progression of cancer [21
]. There is an association between EMT and stem cell transformation in cancers which has been implicated in development of resistance to chemo-therapeutic agents [23
]. Two thirds of the cancer-related deaths in the US involve bone metastasis [24
]. Bone is a favored site of PCa metastasis. The epithelial PCa cells undergo EMT changes which confer invasive potential and lead to metastasis. When metastatic PCa cells reach within the bone milieu, they trigger a cascade of molecular events that produce osteolytic and/or osteoblastic phenomena; and thus, obliteration of EMT alterations at the molecular level has the potential to prevent PCa metastasis.
In the present study we found transformation of the cuboidal epithelial morphology of the PCa cells into the elongated mesenchymal type upon microscopic examination of the tissue sections. Our findings are consistent with previous studies [5
] emphasizing the loss of ‘top-bottom’ (apical-basal) polarity that normally limits the movement of epithelial cells. Due to this morphological change, the cancer cells loose their ability to adhere to the adjacent cells, eventually resulting in cells that are much more motile [26
]. The morphological changes of EMT are accompanied by alterations in the signaling pathways manifested by changes in protein expression. The altered expression levels were prominent at the invasive tumor front (ITF) than at the center of the cancer cell clusters. Our findings are consistent with previous studies [27
]. The localization of EMT at the ITF is important as most aggressive cells and many crucial molecular interactions that enhance or inhibit tumor progression, occur at the tumor-host interface [29
]. Due to EMT molecular alterations, the PCa cancer cells become discohesive at the ITF, migrate out of the tumor cell clusters, travel through lymphovascular system to metastasize to bone [30
]. Once the metas-tatic PCa cells reach the bone, the EMT adhesion molecules re-express by a counter phenomenon referred to as Mesenchymal-to-Epithelial Transition (MET) wherein there is a reversal of the epigenetic mechanism of gene silencing promoting an epithelial phenotype [30
In this study we observed altered expression of EMT markers E-cadherin, Vimentin, PDGF-D, NF-κB, Notch-1 and ZEB1 in PCa and bone metastasis tissues. The over-expression of Notch-1 was statistically significant in bone metastasis as compared to primary PCa (p=0.057). The expression levels, intensity and % of positive cells of the remaining markers were not statistically significant in PCa vs. PCa bone metastasis perhaps due to limited number of cases, suggesting that this proof-of-concept study warrants further in-depth investigation. Notch is a type 1 transmembrane protein which plays a key role in many fundamental cellular processes such as proliferation, stem cell maintenance and differentiation during embryonic and adult development [31
]. Our findings are consistent with previous studies demonstrating Notch-1 over-expression contributing to PCa invasion [32
], metastases [18
] and osteoblastic bone metastases and osteoblastogenesis of human mesenchymal stem cells [33
]. In the bone microenvironment, PCa metastatic cells acquire osteoblastic properties through independent activation of Notch signaling [34
]. Notch-1 and its related genes can serve as novel targets for PCa therapy.
In summary, EMT is a crucial step in the progression and metastasis of PCa. The aberrant expression and localization of E-Cadherin, Vimetin, PDGF-D, NF-κB, Notch-1 and ZEB1, appears to be is important in PCa invasion and bone metastasis. These have the potential to serve as molecular markers of signaling pathways involved in EMT. Most EMT changes were seen at the invasive tumor front indicating the localization of this transient process at the TIF. Notch-1 up-regulation appears to play a significant role in PCa bone metastasis. These preliminary results offer great promise in the clinical arena. However, further validation in a larger cohort of PCa patients are needed to define the cross-talk between EMT signaling pathways in PCa and bone metastasis before these potential molecular markers of EMT can have clinical and translational relevance in the era of personalized medicine.